Mutations caused by activation-induced cytidine deaminase

ABSTRACT

Methods for causing mutations in genes expressed in eukaryotic cells are provided. The methods involve expressing an activation-induced cytidine deaminase (AID) in the cells. The mutated genes can be any gene that is operably linked to a promoter, where the gene is within about 2 kilobases of the promoter. Examples include antibody genes. Also provided are cells expressing AID. The cells can be from any eukaryote, and include hybridoma cells and myeloma fusion partners.

BACKGROUND

(1) Field of the Invention

The present invention generally relates to methods and compositions forinducing mutations in genes in living cells. More specifically, theinvention relates to the use of activation-induced cytidine deaminase toinduce mutations in genes expressed in eukaryotic cells.

(2) Description of the Related Art

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During the normal antibody response, B-cells initially express a highlydiverse repertoire of IgM antibodies that have a low affinity forantigen and are unable to compete with cellular receptors and neutralizeviruses or toxins. In order to make higher affinity antibodies, allvertebrates have evolved mechanisms to introduce mutations into the Vregions of antibody genes that encode the antigen-binding site. Thisprocess is referred to as somatic hypermutation (SHM). The rates of Vregion mutation during this process are 10⁻⁵-10⁻⁴/base pair/generation,which is many orders of magnitude higher than the rates of mutation ofother genes. Hot spot motifs that are preferentially targeted formutation are concentrated in the complementarity determining regions(CDRs), or hypervariable regions, of the V region that encode theantigen binding site. Some of these base changes result in amino acidchanges that increase the affinity and/or change the specificity of theantibody. In man and mouse, V region hypermutation occurs in centroblastB-cells in the germinal centers of secondary lymphoid organs. TheB-cells with higher affinity compete effectively for antigen, present itto T cells and are stimulated to proliferate and differentiate andbecome the majority population in the germinal center. At the same time,in a process called class switching or isotype switching, these B-cellscan rearrange the heavy chain V region gene from its initial locationupstream from the μ constant region to the downstream γ, α, or εconstant regions. This allows the same antigen binding site to beexpressed with each of those isotypes, to carry out the full panoply ofantibody functions and to be distributed throughout the body. During thecourse of the primary antibody response, these mutational and isotypeswitching events begin to occur around 7 days after immunization andcontinue until about 10 days, after which these B-cells cease mutatingand isotype switching and leave the germinal center to differentiateinto plasma cells and memory B-cells.

The molecular and biochemical mechanisms responsible for V regionhypermutation and isotype switching are poorly understood. However, inthe last few years a few of the enzymes involved in both processes havebeen discovered. One of these is activation-induced cytidine deaminase,or AID, which is normally expressed exclusively in germinal centerB-cells (Muramatsu et al., 1999). Mice genetically defective in AID anda subset of hyper-IgM patients with mutations in AID do not undergo SHMor isotype switching.

AID has 50% amino acid homology with APOBEC, which is an RNA editingcytidine deaminase that creates a stop codon in the mRNA forapolipoprotein B (apoB). This results in a truncated protein with achange in function that is required for the normal function of apoB. Itis not known whether AID in B-cells acts directly on the DNA of the Vregion gene to produce mutations or as an RNA editing enzyme to activateproteins that are required for V region mutation and isotype switching(Kinoshita and Honjo, 2001).

Recent studies have shown that lowering the level of expression of AIDby as little as 5-fold is associated with loss of V region mutation incultured germinal center Ramos Burkitt's lymphoma B-cells (“Ramoscells”)(Zhang et al., 2001). However, there are no published studiesthat establish that overexpressing AID in those cultured B-cells thathad lost the ability to mutate turns on mutation again at the wild typerate. There are also no published studies indicating that expressing AIDin plasma cells or in non-B-cells turns on the SHM process.

Monoclonal Antibodies

Monoclonal antibodies have become valuable research and therapeutictools. They are routinely generated by fusing antibody forming B-cellsfrom animals or humans to continuously growing myeloma or othermalignant B-cells. The resulting “hybridomas” produce monoclonalantibodies having homogeneous binding sites that are used as scientificand manufacturing reagents and in the diagnosis, prevention andtreatment of disease. Monoclonal antibodies can also be generated byimmortalizing B-cells with viruses (e.g., EBV) or by expression ofoncogenes, or transfecting immunoglobulin genes into already establishedcell lines.

Six monoclonal antibodies have been approved by the FDA for therapeuticuse and more than 60 others are in clinical trials. In addition, manyother mouse and human monoclonal antibodies are currently being used asresearch and diagnostic reagents, including the production ofmacromolecules. New monoclonal antibodies are being routinely generatedin numerous laboratories.

One of the persistent problems with the current state of hybridomatechnology is that most monoclonal antibodies produced are of lowaffinity (i.e., they bind antigen relatively weakly). This is becauserelatively synchronized, early blasting B-cells are the cells that aremost likely to form viable hybrids with the cultured myeloma cells. TheB-cells that arise early in the primary or even the secondary responsehave not undergone as many rounds of somatic mutation, and more highlymutated B-cells are only rarely captured as hybridomas during the fusionprocess. In addition, some potentially useful monoclonal antibodies havecross-reactivities with self-antigens, or other cross-reactivities thatmake them less useful. The specificity to the antigen used to induce theantibody may also be undesirably high (i.e., does not bind to epitopesthat are very similar, to which binding is desired) or undesirably low(i.e., binds excessively to similar epitopes).

Hybridoma cells that make monoclonal antibodies are plasma cells, whichnormally undergo very low rates of V region mutation. Mutants ofhybridoma cells that arise in culture are almost all deletions in theconstant region (Kobrin et al., 1990), and the few variable regionmutants that have been identified arise at frequencies lower than 10⁻⁶(Id.).

Previous studies have established that hybridomas can be switched inculture to express other isotypes. The frequency of such class switchescan be increased by selecting for higher switching subclones (Muquan etal., 1996). Additionally, cultured hybridoma cells transfected with Iggenes can support rates of mutation that have been recorded at10⁻⁴-10⁻⁵/bp/gen (Green et al., 1998). However, those transfected cellscontained multiple copies of the Ig gene, and the recorded rate was fora single nonsense mutation that was embedded in a hot spot for mutation.We now estimate that the overall rate of mutation of the average base inthe V region in those studies was 20-100 fold lower. Nevertheless, thosestudies do show that hybridoma cells can undergo rather high rates of Vregion mutation. In addition, if one fuses a non-mutating hybridoma(e.g., NSO—the fusion partner) to a mutating pre-B-cell (18-81), somehybrids have high rates of mutation (Green et al., 1997). Stable highlymutating clones can also be isolated (Id.). However, 18-81, which hasrecently been shown to express AID, is the only pre-B-cell that has everbeen shown to mutate constitutively in culture. However, no publishedstudies have suggested that AID is the sole factor required to inducehigh rates of mutations in the B-cells or hybrid cells, or that antibodygenes in any hybridoma cell culture can be made to undergo high rates ofmutation. The literature on mutation in plasma cells in culture isreviewed in Kobrin et al., 1990 and Green et al., 1998.

In many cases, if an existing monoclonal antibody could be altered toproduce a higher affinity towards a specific antigen, it would be moreeffective and could be used in smaller amounts, thus reducing its cost.Higher affinity monoclonal antibodies would be especially useful totherapeutically target tumors (Zuckier et al., 2000) or neutralizeviruses or toxins that bind to high affinity cellular receptors. Higheraffinity monoclonal antibodies would also be useful in the preventionand treatment of infection with viruses such as Ebola and Lhasa Fever,or other agents that could be used as germ warfare agents. High affinitymonoclonal antibodies could also be used against a variety of toxinssuch as botulinus and ricin for similar purposes.

In some cases, diagnostic or therapeutic monoclonal antibodies havecross-reactivity to a self antigen, which can produce toxicity orinterfere with a diagnostic assay. If random mutation of the bindingsite were possible, variants without that cross reactivity could beidentified and isolated.

The generation of monoclonal antibody class switching would also beuseful. IgGs have a longer half-life than IgM and penetrate tissues andthe placenta better. Human IgG1, 2, and 4 have half-lives of 25 dayswhile IgG3 has a half life of only 7 days (Zuckier et al., 1998). Longor short half-lives have different benefits in different situations,making one or the other of these isotypes more useful. Also, IgAantibodies are particularly resistant to gut proteases. For all of thesereasons, the ability to induce high rates of somatic mutation andisotype switching in vitro would make monoclonal antibodies more useful.The present invention satisfies that need by providing methods andcompositions for inducing SHM in antibody genes in hybridomas as well asin other proteins in other cells.

SUMMARY OF THE INVENTION

Accordingly, the inventors have discovered that the expression ofactivation-induced cytidine deaminase (AID) in eukaryotic cells causesincreased rates of mutation of genes expressed in the eukaryotic cells,when the genes are operably linked to a promoter, and when the promoteris within about two kilobases of the gene. Based on that discovery,methods and reagents are provided to routinely create mutations in anygene that can be expressed in eukaryotic cells, including in particularantibody genes in monoclonal antibody-producing hybridomas. AIDexpression can also cause isotype class switching.

Thus, in some embodiments, the present invention is directed to methodsof inducing a mutation in a gene in a eukaryotic cell, where the gene isoperably linked to a promoter, and where the gene is within about twokilobases of the promoter. The methods comprise expressing a transgenicactivation-induced cytidine deaminase (AID) gene in the cell.

In other embodiments, the invention is directed to methods ofdetermining the effect of mutations in a gene encoding a protein on thephenotype of the protein in a eukaryotic cell. In these methods, thegene is operably linked to a promoter, and is within about two kilobasesof the promoter. The methods comprise expressing the protein and atransgenic AID gene in the eukaryotic cell, establishing clonal coloniesof the cell, identifying clonal colonies that produce a gene of theprotein that has a mutation, determining whether the protein expressedby the mutated gene in any clonal colonies identified has an alteredphenotype, and associating the altered phenotype with a particularmutation.

Additionally, the invention is directed to methods of inducing amutation in an antibody gene in a eukaryotic cell. The methods compriseexpressing a transgenic AID gene in the cell.

The present invention is also directed to methods of inducing a classswitch in an antibody gene in a eukaryotic cell. The methods compriseexpressing a transgenic AID gene in the cell.

In additional embodiments, the invention is directed to methods ofaltering an affinity or a specificity of a monoclonal antibody to anantigen, or altering a cross-reactivity of the monoclonal antibody to asecond antigen. In these methods the monoclonal antibody is produced bya eukaryotic cell that is capable of expressing a transgenic AID geneunder inducible control. The methods comprise expressing the AID gene inthe eukaryotic cell for a time and under conditions sufficient to inducea mutation in a gene encoding the monoclonal antibody, suppressingexpression of the AID gene in the eukaryotic cell, establishing clonalcolonies of the cell, and determining whether the monoclonal antibodyproduced by any of the clonal colonies of the cell has altered affinityor specificity to the antigen, or altered cross-reactivity to the secondantigen.

The present invention is additionally directed to various eukaryoticcells that comprise an AID gene. In some of these embodiments, the AIDgene is transgenic. In those eukaryotic cells, expression of the AIDgene is preferably inducible. Cells envisioned in these embodimentsinclude myeloma fusion partners and hybridomas that express an AID gene.In other embodiments the eukaryotic cells expressing the AID gene arenot B-cells.

The invention also encompasses the mutated genes produced by theabove-described methods and cells, the proteins encoded by those mutatedgenes, and cells that comprise those genes or proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes experimental results which establish that human AID(HAID) expression induces SHM in non-mutating Ramos cells. RNA wasisolated from (a) selected subclones of Ramos cells (Zhang et al., 2001)and (b) stable transfectants of the non-mutating Ramos clone 1 thatoverexpress hAID. HAID and GAPDH were reverse transcribed, and five folddilutions of the resulting cDNA were amplified by PCR. Levels of AIDmRNA in clones 6 and 7 are about 5-fold higher than in clone 1 (Zhang etal., 2001) (Panel a), and are ˜25 fold higher in clones A.2 and A.5 thanin C.1 and A.1 (Panel b). The mutation rates shown for the subclones inPanel a are taken from Zhang et al., 2000, and in Panel b are from thepresent work.

FIG. 2 summarizes experimental results which show the induction of SMHin hybridomas transfected with AID. Panel a. Hybridomas N89 (nonsense inleader) and N114 (nonsense in V-region) were transfected with emptyvector or the hAID construct. Frequency of nonsense revertants, asdetected with the ELISA spot assay (Spira et al., 1993), is plotted.Typical ELISA spots for N89 are shown in inset. The difference in thefrequencies of reversion of N114 vector and N114 hAID was statisticallysignificant (P<0.05). Panel b. Northern blots for hAID and GAPDH of N89,N114, and P1-5 transfected hybridoma clones. Clone numbers for N89 andN114 clones correspond to numbers in Panel a.

FIG. 3 summarizes mutation data observed in hybridoma clones. Panel a.Pie charts, as previously shown (Sale and Neuberger, 1998), depictingthe distribution of the frequencies of mutation of P1-5 and N114hybridoma clones. Shown are the number of sequences analyzed (center ofpie) and the proportion of sequences with 0, 1, 2 . . . mutations (pieslices). Panel b. All mutations located within the V-region (V186.2) ofhybridoma P1-5 clones are shown. Duplicate mutations were counted oncein Table 1, unless genealogies indicate mutations were unique. Hotspotmotifs (RGYW and WRCY) are bolded. Although other hot spot motifs arefrequently mutated, we and others have observed a high frequency ofmutation at the codon 31 hotspot (underlined) in vivo (Sack et al.,2001).

FIG. 4 summarizes results of experiments establishing that AID inducesmutations in cells other than B-cells, here T cells (Bw-5147) andChinese hamster ovary cells (CHO). CHO and Bw5147 cells werestably-transfected with the same immunoglobulin heavy and light chaingenes used previously (Lin et al., 1998). The heavy chain gene (Igγ2a)has a nonsense codon within the V-region, and thus cells cannot producefunctional IgG2a unless the nonsense codon is mutated. These cells weretransfected with empty vector (open circles) and vector expressing hAID(filled in circles). The ELISA-spot assay was used to assay for cellssecreting IgG2a that have reverted the nonsense codon of the heavy chaingene. Because some clones transfected with hAID failed to express AID(see lower panel: Northern blots for AID and GAPDH), the data pointsfrom such clones were placed with the empty vector-transfected clones.Thus, the columns are divided in clones that are AID-negative and clonesthat are AID-positive. Analysis of the primary data by the independentsamples t-test (with equal variances assumed) shows that the reversionfrequencies between the AID−ve and AID+ve are statistically significant(p<0.05 and p<0.01 for Bw5147 and CHO, respectively).

FIG. 5 summarizes results from experiments establishing that AIDhypersensitizes Ramos cells to class-switch recombination. IndicatedRamos clones were incubated with empty-vector-transfected NIH3T3 cells(− stimulation) or with CD40L-transfected NIH3T3 cells and 5 ng/ml ofIL-4 (+ stimulation) for 10 days. Panel A shows results of ELISA testingof supernatants for secreted IgG and IgM. Panel B shows results ofRT-PCR analysis for IgG mRNA and the sterile transcripts Iγ1, 2, 3 on10-day stimulated and unstimulated clones.

FIG. 6 summarizes results from experiments showing mutations in the AIDtransgene from Ramos, hybridoma P1-5, and CHO cells. Mutations locatedwithin the AID transgene from the Burkitt's lymphoma Ramos (upper case),hybridoma P1-5 (lower case), and CHO cells (upper case bolded).Duplicate mutations from each clone were counted once in Table 3.Hotspot motifs (RGYW and WRCY) are bolded.

FIG. 7 summarizes experimental results establishing that AID induces SHMin CHO cells. Panel A. Murine Vn/ECMV γ2a-construct transfected into CHOcells to study SHM. Previously described in Lin et al., 1998, this heavychain immunoglobulin construct has replaced the intronic μ enhancer witha CMV enhancer, and contains a TAG nonsense codon within an RGYWhot-spot motif at codon 38. Panel B. Left two columns: CHO cloneCHO-LC18 (see Materials and Methods) stably transfected with heavy andlight chain immunoglobulin genes was transfected with empty vector (opencircles) or the hAID construct (filled in circles). Depending onexpression of AID (see below), data was distributed into AID-negative(AID−) and AID-positive (AID+) columns. Frequency of nonsenserevertants, as detected with the ELISA spot assay, is plotted. Right twocolumns: 10 subclones of CHO clones A.3 and A.9 were further analyzedusing the ELISA spot assay to calculate mutation rates by fluctuationanalysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the expression ofactivation-induced cytidine deaminase (AID) in eukaryotic cells causesincreased rates of mutation of genes expressed in the cells, when thegenes are operably linked to a promoter, and when the promoter is withinabout two kilobases of the gene. This discovery makes possible thedevelopment and use of methods and reagents to routinely createmutations in any gene that can be expressed in eukaryotic cells,including native genes and genes introduced into the celltransgenically. In particularly useful embodiments, expression of AID inmonoclonal antibody (Mab)-producing hybridomas causes high rates ofmutation and class switching in the antibody genes, allowing theselection of monoclonal antibodies with altered affinity, specificity,cross-reactivity to an antigen, or isotype.

It has also been discovered that the mutating effects of AID on a genecan be negated by flanking the gene, either at the 5′ or the 3′ end,with foreign sequences (i.e., sequences not native to the cell). See,e.g., Example 5. In these embodiments, the flanking sequence is at leastabout 200 bp, more preferably at least about 1000 bp, and mostpreferably at least about 2000 bp. In the most preferred embodiments,the foreign sequence flanks both the 5′ and the 3′ end of the gene. Itis also preferred that the foreign sequences are sequences are from aspecies that is highly unrelated to the cell, e.g., yeast sequences whenthe cell is a mammalian cell. In the most preferred embodiments, thesequences are bacterial (e.g., E. coli) sequences.

This discovery is particularly useful for creating cells or organismscomprising a transgenic AID gene that is not subject to mutation by itsown gene product. Thus, by retaining the parts of a plasmid vector whichhas foreign (e.g., bacterial) DNA sequences flanking the AID gene, andstably integrating those foreign sequences with the AID gene into thehost cell, AID can be introduced and overexpressed in a way thatprevents the AID gene product from mutating the introduced AIDtransgene.

Activation-induced cytidine deaminase (“AID”) genes useful for themethods and compositions of this invention can be any vertebrate AIDgene, defined herein as a cytidine deaminase that is naturally inducedupon activation of B-cells in the vertebrate. In preferred embodiments,the AID gene is a mammalian AID gene, as exemplified in GenBankaccession numbers NM020661 (human) or NM009645 (mouse). In the mostpreferred embodiments, the AID gene is a human AID gene.

As used herein, “mutation” refers to an alteration in a basepair of agene (also known as a point mutation, e.g., C to T) or the alteration inthe amino acid sequence of a protein as a result of the alteration inthe gene sequence. The gene mutation can cause the generation of apremature stop codon in the gene, causing a truncated protein, or noprotein, to be synthesized.

As used herein, the terms “gene expression” and “protein expression” aresynonymous and refer to the transcription and translation of a gene intoa protein encoded by the gene.

According to the present invention, expression of AID in a eukaryoticcell causes mutations in the eukaryotic cell. This discovery allows thedevelopment of methods of inducing a mutation in a gene in a eukaryoticcell. The methods comprise expressing a transgenic activation-inducedcytidine deaminase (AID) gene in the cell. In these methods, the genethat is subject to mutation is any gene that is operably linked to apromoter, where the gene is within about two kilobases (kb) of thepromoter. See Rothenfluh et al., 1994; Rada and Milstein, 2001. Inpreferred embodiments, the gene is also operably linked to an enhancer,since enhancers increase expression of the gene to the high levelsneeded to achieve measurable mutation by the expressed transgenic AID.The gene is also preferably between 10 bases and 2 kb in the 3′direction from the promoter. See Wu and Claflin, 1998. See also Example1 and FIG. 2, where some of the mutations generated targeted very nearthe start of transcription.

As used herein, a transgenic gene or a transgene is a gene that ispresent in a cell due to molecular genetic manipulation. The gene can beintegrated into the genome or the cell or present in the cellextrachromosomally, e.g., as part of a plasmid or virus. The gene can bestably maintained in the cell or transiently maintained, then lost fromthe cell.

In the methods of the present invention, the mutation of the gene causedby AID expression in the cell is an average mutation rate in the genethat is at least twice that of the mutation rate of that gene withoutAID. In preferred embodiments, the mutation rate is at least 5 times,more preferably 10 times, still more preferably 50 times the averagemutation rate of the gene in the cell not expressing AID. In the mostpreferred embodiments, the average mutation rate is at least 100 timesthe average mutation rate of the gene in the cell not expressing AID.

In the methods of this invention, AID-induced mutation occurs morefrequently where the nucleic acid sequence of the gene corresponds tothe well known “hot spot” sequences of variable regions of antibodygenes. Those hot spots have the sequences are RGYW or WRCY (R=A or G,Y=C or T, W=T or A). Thus, genes that have higher incidences of thesehot spot sequences would be expected to have higher mutation rates thanthose genes that have fewer hot spot motifs. Thus, mutation rates at anyparticular basepair, particularly at the G of an RGYW motif, or a C of aWRCY motif, can be 1000 times, or more, the mutation rate at thatbasepair in the absence of AID.

Any promoter or enhancer known in the art that allows the expression ofthe gene in the eukaryotic cell can be used for these methods.Preferably, the promoter allows moderate to high expression of the genein the cell. The amount of expression can be measured by any means knownin the art, including quantitative measurement of the gene product, orpreferably quantitation of polyA mRNA. A useful measurement of geneexpression of a particular gene is the determination of the relativeamount of polyA mRNA of the gene compared to total mRNA in the cell. Theskilled artisan can make this determination without undueexperimentation using well-known methods. In preferred embodiments, thegene to be mutated comprises at least 0.01% of total polyA mRNA in thecell. In more preferred embodiments, the polyA mRNA of the genecomprises at least 0.1% of total polyA mRNA in the cell. In still morepreferred embodiments, the polyA mRNA of the gene comprises at least0.5% of total polyA mRNA in the cell. In the most preferred embodiments,the polyA mRNA of the gene comprises at least 1% of total polyA mRNA inthe cell. For B-cells, a preferred promoter is an immunoglobulinpromoter and, where present, a preferred enhancer is an immunoglobulinenhancer. For other mammalian cells, preferred promoters and enhancers(where present) are viral promoters and enhancers, many suitableexamples of which are known in the art.

In these methods, the AID gene can be expressed constitutively in thecell. In preferred embodiments, however, the AID gene expression isinducible. Inducible AID expression is preferred because this allows thegeneration of mutants when AID is expressed, and then the selection andevaluation of the generated mutants when AID is not expressed, thusavoiding the possibility of the further generation of mutants during theselection and evaluation steps, or at other times when mutation is notwanted.

In embodiments where the AID gene is inducible, the system used tocontrol induction is not narrowly limited, and can be selected by theskilled artisan without undue experimentation based on particularcharacteristics of the cell type and gene in which mutation is desired.Among the preferred induction systems for mammalian cells is thewell-known positive or negative regulatory tet system (Gossen andBujard, 1992; Gossen et al., 1995) and the ecdysone receptor-induciblesystem (See, e.g., No et al., 1996; Albanese et al., 2000).

Because the accessory proteins and enzymes associated with SHM, i.e.,MSH2, MSH6, and polymerases ζ and η are present in all eukaryotic cells(see, e.g., Bowers et al., 2000; Nelson et al., 1996; Washington et al.,2001, describing these proteins and enzymes in yeast), it is expectedthat any eukaryotic cell can be utilized in these methods. Included areyeast and plant cells. In preferred embodiments, the eukaryotic cell isa vertebrate cell. In more preferred embodiments, the cell is amammalian cell, including mouse, rat and human cells. Any eukaryoticcell type that can be cultured is expected to be useful for thesemethods. See also Example 2, where gene mutation is induced in Chinesehamster overy (CHO) cells and T cells expressing an AID transgene. Insome preferred embodiments the cell can be a B-cell, for example ahybridoma expressing an antibody gene to which mutation is desired.

In these methods, the gene subject to mutation can be a native gene(i.e., a gene in its natural chromosomal or extranuclear location in thecell), provided that the gene is operably linked to a promoter and,preferably, an enhancer, and the gene is within about 2 kb of thepromoter.

Alternatively, the gene subject to mutation can be a transgeneintroduced into the cell transiently or stably, and integrated into thegenome or present in an extranuclear vehicle such as a plasmid or avirus. The gene can also be of prokaryotic or eukaryotic origin, e.g.,from a microbe, plant, insect, vertebrate, etc., including a mammal or ahuman. It is well established that any gene can be mutated by SHMmechanisms if properly positioned and operably linked to a promoter and,preferably, an enhancer. See, e.g., Peters and Storb, 1996; Yelamos etal., 1995; Tumas-Brundage and Manser, 1997; and Shen et al., 1998.Martin and Scharff (2002), provided herein as Example 4, furtherconfirms the expectation that non-immunoglobulin genes, including theAID transgene itself, are subject to AID-induced SHM, both in B cellsand non-B cells.

These methods of causing a mutation in a gene can be used, for example,to determine the effect of the generated mutations in the structure orfunction of the protein encoded by the gene. The methods can also beused to create mutants of a protein that has desirable alteredcharacteristics. Nonlimiting examples include mutants of bindingproteins such as antibodies, cytokines or transcription factors, wherethe mutants have altered specificity or affinity or block the effect ofthe binding protein; mutants of enzymes, where the mutants have alteredcatalytic activities or environmental optima; mutants of toxins, wherethe mutants have altered toxicity or antitoxin activity; and mutants ofstructural proteins, where the mutants affect cellular or tissuemorphology.

Thus, the present invention is also directed to methods of determiningthe effect of mutations in a gene encoding a protein on the phenotype ofthe protein in a eukaryotic cell. As in the previously describedmethods, the gene to be mutated must be operably linked to a promoterand an enhancer, and within about two kilobases of the promoter. Themethods comprise the following steps:

-   -   (a) expressing the protein and a transgenic AID gene in the        eukaryotic cell;    -   (b) establishing clonal colonies of the cell;    -   (c) identifying clonal colonies that produce a gene of the        protein that has a mutation;    -   (d) determining whether the protein expressed by the mutated        gene in any clonal colonies identified in step (c) has an        altered phenotype; and    -   (e) associating the altered phenotype with a particular        mutation.

The above steps need not be performed in the order set forth.

As with the previously described methods, these methods can employ anyeukaryotic cell that can be cultured, and any gene from any source. Anypromoter and enhancer (when employed) can also be used, but preferredare those that allow moderate to high expression of the gene. InducibleAID expression is preferred, e.g., using a let or ecdysone receptorsystem, so that AID expression can be induced only during step (a) toavoid generation of mutants during the subsequent steps.

For these methods or any other methods described herein, theidentification of mutants as in step (c) can be by any means suitablefor the gene and cell type involved. In some embodiments, the entiregene from each clonal colony can be sequenced, e.g., after PCRamplification. Alternatively, only a portion of the gene can besequenced, for example the portion encoding the active site of an enzymeor a binding protein.

In other embodiments of these methods or other invention methods, thecolonies harboring mutant genes can be identified by changes in theexpressed mutant protein encoded by the gene, or, where appropriate,changes in cell or colony phenotype engendered by the mutation. Thoseclones expressing the desired phenotype are then sequenced to determinethe mutation that is causing the phenotypic change. In the presentmethods, this is equivalent to performing step (d) before step (c). Theclonal colonies can be screened, for example, for visible changes in thecolony or cell morphology, or for changes in binding of antibodies tothe protein, such as an elimination, reduction, increase, orcommencement of antibody binding. A useful assay for screening withantibodies is the ELISA spot assay (Spira et al. 1993).

For these methods or any other methods described herein, once the targetgene is induced to undergo reasonably high rates of mutation (and/orisotype switching—see below) in tissue culture, there are manytechniques that allow even relatively rare subclones expressing adesired protein to be separated from the rest of the cells and thenpropagated to produce the mutant protein. When the protein is anantibody, useful methods include enrichment of cells producing highaffinity antibodies by FACS after staining with limiting amounts offluorescent-labeled antigen or tetramers, use of antigen coated beads toenrich for higher affinity subclones and sib selection using the ELISAspot assay to screen for variants. Similar techniques using anti-isotypespecific antibodies are routinely used to isolate isotype switchvariants (Spira et al., 1993).

The above methods are particularly useful for inducing mutations inantibody genes. Thus, some embodiments of the invention are directed tomethods of inducing a mutation in an antibody gene in a eukaryotic cell.The methods comprise expressing a transgenic AID gene in the cell. Aswith previous methods, the antibody gene can be native to the cell, forexample as in a hybridoma cell. Alternatively, the antibody gene can bea transgene in any eukaryotic cell, such as mammalian cells (e.g., CHOor T cells—see Example 2), yeast cells, plant cells, insect cells,vertebrate cells, etc. The antibody gene can be from any vertebratespecies, for example, rat, mouse, rabbit, hamster, or human.

The antibody gene can be genetically unaltered before the AID gene isexpressed, i.e., a heavy or light chain gene as are naturally made inB-cells. Alternatively, the antibody gene can be altered by any meansknown in the art, for example as with humanized antibodies (Vaswani andHamilton, 1998), single chain antibodies and fragments (Fischer et al.,1999; Worn and Pluckthun, 2001) and multivalent antibodies (see, e.g.U.S. Pat. No. 6,121,424).

As in previous embodiments, these methods can utilize constitutivecontrol of AID gene expression in the cells. However, inducible control,e.g., using a let or ecdysone receptor system, is preferred.

For most practical purposes, it is preferred that the antibody gene inthese embodiments encode at least a portion (e.g., a light chain or aheavy chain) of an antibody that binds to an antigen. Both light chainand heavy chain antibody genes can also be mutated.

The methods of the invention are not limited to the mutation of antibodygenes encoding antibodies that bind to any particular antigen. Forexample, the mutated antibody gene can encode at least a portion of acatalytic antibody (i.e., an antibody that catalyzes a chemical reaction[Wentworth and Janda, 1998]). The mutated antibody gene can also encodeat least a portion of an antibody that binds to a pathogen, for examplean animal pathogen, e.g., a human pathogen. The pathogen can be abacterium, virus, or any other organism. The antigen can also be atoxin, such as polypeptide toxins produced by microorganisms or plants(e.g., ricin). The antigen can also usefully be an enzyme, atranscription factor, a cytokine, a structural protein, or any otherprotein. Antibodies to any macromolecules such as carbohydrates, nucleicacids, lipids, and small chemicals such as haptens are also envisionedas benefitting from these methods.

The mutant antibody produced as a result of these methods can have anyof a number of alterations in its antigen binding capacity. It can havehigher or lower affinity for the antigen than before the mutation. Itcan also have higher or lower specificity for the antigen than beforethe mutation. Additionally, it can have altered cross-reactivity (eitherincreased or decreased) for a second antigen than before the mutation.

As is well known, AID is required for both antibody class switching andsomatic hypermutation in activated B-cells. However, there are nopublished studies showing that expression of AID in plasma cells or inB-cells at other stages of differentiation induces class-switchrecombination. This is established in Example 3, which shows spontaneousclass-switching caused by expression of AID. Therefore, the provision ofAID in a eukaryotic cell expressing antibody heavy chain genes inducesclass switching if the genes of alternate classes are also present inthe same configuration as those genes are present in a B-cell. Theinvention is thus directed to methods of inducing a class switch in anantibody heavy chain gene in a eukaryotic cell, the method comprisingexpressing a transgenic AID gene in the cell.

As with previous methods, it is preferred that the AID gene is underinducible control (e.g., with a tet system), although constitutivecontrol is also envisioned. Also as with previous methods, anyeukaryotic cell can be utilized in these methods, provided the cellharbors both the antibody gene and at least one gene for the isotype towhich the switch is desired, in the B-cell configuration required forclass switching. In preferred embodiments, the cell is a myeloma cell,most preferably a hybridoma cell, since those cells already makeantibody genes and comprise properly configured alternative classes.

As with previous methods, antibodies from any species, as well asgenetically altered (e.g., humanized) antibodies can be employed inthese methods. Also, the methods are not narrowly limited to antibodieshaving any particular antigen specificity, and includes catalyticantibodies, antibodies to pathogens or toxins, or antibodies to haptens,enzymes, transcription factors, cytokines, and structural proteins.

In related embodiments, the present invention is directed to methods ofaltering an affinity or a specificity of a monoclonal antibody to anantigen, or altering a cross-reactivity of the monoclonal antibody to asecond antigen. These methods require the monoclonal antibody to beproduced by a eukaryotic cell that is capable of expressing a transgenicAID gene under inducible control. The methods comprise

-   -   (a) expressing the AID gene in the eukaryotic cell for a time        and under conditions sufficient to induce a mutation in a gene        encoding the monoclonal antibody;    -   (b) suppressing expression of the AID gene in the eukaryotic        cell;    -   (c) establishing clonal colonies of the cell; and    -   (d) determining whether the monoclonal antibody produced by any        of the clonal colonies of the cell has altered affinity or        specificity to the antigen, or altered cross-reactivity to the        second antigen.

The preferred cells for these methods are hybridoma cells, although anyeukaryotic cell could be usefully employed. As with previous methods,these methods are not limited to use with antibodies from any particularspecies, or binding any particular antigen.

In some embodiments of these methods, steps (a) through (d) are repeatedwith a clonal colony that has altered affinity or specificity to theantigen, or altered cross-reactivity to the second antigen. This allowsthe generation of clones that produce antibodies with severalaccumulated mutations.

The step (d) selection for particular clones of interest can bedeveloped for any particular antibody by the skilled artisan withoutundue experimentation. For example, where an antibody that has greateror less specificity is desired, the candidate clones can be screenedwith a labeled antigen and antigens of similar structure, eitherseparately, or in competition with each other. Myriad other assays canbe easily developed to select antibodies with increased or decreasedaffinity to the antigen, or increased or decreased cross-reactivity witha second antigen.

In other embodiments, the invention is directed to eukaryotic cellscomprising a transgenic AID gene, wherein expression of the AID gene isinducible. These embodiments are not narrowly limited to any particularinduction system, and any appropriate system can be adopted by a skilledartisan without undue experimentation. For mammalian cells, a preferredsystem is the tet system (either inducible or repressible by doxycyclineor analogs) or an ecdysone receptor system, since these systems affordvery tight regulatory control.

The cells of these embodiments can be any eukaryotic cell, including butnot limited to yeast, insect, vertebrate or mammalian (including human)cells. Among preferred mammalian cells are Chinese hamster ovary (CHO)cells, T cells, or myeloma (including hybridoma) cells.

Since the cells of these embodiments are able to cause mutations inexpressed genes, the cells can further comprise a gene encoding aprotein, wherein the gene is operably linked to a promoter and,preferably an enhancer, and wherein the gene is within about twokilobases of the promoter. In those cells the gene can be a native geneor a transgene. Preferably, the gene undergoes mutation upon expressionof the AID gene. In some preferred embodiments of these cells, the geneis an antibody gene.

The invention is also directed to eukaryotic cells expressing an AIDgene, wherein the cell is not a B-cell. In these cells, the AID gene canbe a native gene, for example when the cells are created by cell fusionbetween a B-cell and a non-B-cell, and the cell derives its expressionof AID from the B-cell. Thus, the cells of these embodiments can behybrid cells that are partially B-cells. Examples of B-cells that can beused for these hybridizations are Ramos cells that express AID. See,e.g., Example 1.

In preferred embodiments of these cells, the AID gene is a transgene. Asin previously described methods and cells, the AID can be constitutivelyexpressed, or inducible, for example using a tet or ecdysone system.

The cells of these embodiments can be any eukaryotic cell as appropriatefor any particular application. Nonlimiting examples include yeastcells, insect cells, and vertebrate cells, including mammalian (e.g.,human) cells. They can also be any type of cell that can be maintainedin culture. Preferred examples include T cells and CHO cells.

As with previously described embodiments, the cells of these embodimentscan also comprise a gene, which can be a native gene or a transgene,operably linked to a promoter and an enhancer, wherein the gene iswithin about two kilobases of the promoter. Preferably, the geneundergoes mutation upon expression of the AID gene. A particularlyuseful example of the gene is an antibody gene.

Related to the above described cells, the invention is also directed toa myeloma fusion partner expressing an AID gene. As used herein, amyeloma fusion partner is a myeloma cell that can be grown in cultureand that has a selection system that allows for the efficient selectionof hybrid cells when the fusion partner is fused with a B-cell duringthe production of hybridomas. A preferred example of a selection systemis a deficiency in HGPRT, which allows selection of the hybridoma onhypoxanthine-aminopterin-thymidine (HAT) media. Examples of commonlyused myeloma fusion partners are Sp2/0-Ag 14, FOX-NY, P3X63, NX-1, P3,P3X643 Ag8.653, NS1, and NSO.

The myeloma fusion partners of these embodiments are useful in theproduction of hybridomas producing monoclonal antibodies that can bemutated when the AID gene is expressed. To produce such hybridomas, thepractitioner need only fuse these fusion partners with B-cells using theusual hybridoma production protocol. Thus, these myeloma fusion partnersallow the mutation of monoclonal antibodies in any hybridoma, withouthaving to transfect the hybridoma with an AID gene.

The AID gene in the myeloma fusion partner can be native, which can becreated by fusing an AID-producing Ramos B-cell with a myeloma fusionpartner that is not producing AID. Preferably, however, the AID gene isa transgene.

The AID gene can be constitutively expressed. However, it is preferredthat the AID gene is inducible, e.g., with tet or ecdysone selection,since those systems allow precise control of when mutations can becreated in hybridomas produced using the cells.

The present invention is also directed to a hybridoma expressing an AIDgene. Such a hybridoma can be created, for example, by fusing a non-AIDexpressing hybridoma with a cell expressing AID, such as a Ramos B-cellor a myeloma. Alternatively, a hybridoma that does not produce AID canbe selected to produce AID. In preferred embodiments, the hybridoma iscreated by transfecting a hybridoma that does not express AID with avector encoding an AID gene. Such vectors can be designed and created bythe skilled artisan without undue experimentation. In other preferredembodiments, the hybridoma expressing AID is created by fusing a B-cellwith the myeloma fusion partner previously discussed that is capable ofexpressing a transgenic AID gene. Thus, although the AID gene in thesehybridomas can be native, it is preferred that it is transgenic. It isalso preferred that AID gene expression be inducible in thesehybridomas, although constitutive expression is also envisioned.

In preferred embodiments to these hybridoma cells, the hybridomaexpresses an antibody that binds to an antigen. These embodiments arenot limited to hybridoma cells expressing antibodies to any particularantigen, nor from any particular species.

In some embodiments of these hybridoma cells, the antibody geneexpressed therein has undergone mutation upon expression of the AID geneto cause a mutation in the antibody. The mutation can cause a change inthe antibody affinity or specificity to an antigen, or thecross-reactivity of the antibody to a second antigen. Alternatively, themutation can cause no discernable change in the antibody bindingcharacteristics. This lack of discernable change can be due to themutation altering an amino acid residue that does not affect theantibody binding characteristics. The lack of change can also be due tothe mutation being silent by changing a nucleotide residue that has noeffect in the amino acid sequence due to the redundancy of the geneticcode.

The antibody produced by the hybridomas in these embodiments can alsohave undergone a class switch during AID gene expression, with orwithout mutation in the antibody.

Other embodiments of the present invention includes mutated genes,including antibody genes, produced by any of the above methods; mutatedproteins (including antibodies) encoded by any of those mutated genes;mutated genes and proteins produced by any of the above described cells;vectors useful for producing any of the above-identified cells; andeukaryotic cells comprising any of the mutated genes produced by theabove methods or cells.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols inMolecular Biology” Volumes I-IV (Ausubel, R. M., ed. (1997); “CellBiology: A Laboratory Handbook” Volumes I-III (J. E. Celis, ed. (1994);and “Current Protocols in Immunology” Volumes I-III (Coligan, J. E., ed.(1994).

EXAMPLE 1 Expression of AID and Somatic Hypermutation of Antibody Genes

In this Example, we establish that AID is required for somatichypermutation (SHM) in centroblast-like Ramos cells. We then show thatexpression of AID is sufficient to induce SHM in hybridoma cells, whichrepresent a later stage of B-cell differentiation that does not normallyundergo SHM. Methods.

Cell lines, cell culture and transfection conditions: Ramos cells weregrown as previously described (Zhang et al., 2001). N89 and N114hybridoma cells were described previously (Connor et al., 1994), whilethe hybridoma P1-5 was obtained from Dr. Alfred Bothwell (Tao andBothwell, 1990). Ramos cells were electroporated with 10 μg linearizedDNA in IMDM medium at 250 volts, 960 μF, plated into 96 well plates, andselected with 0.6 mg/ml hygromycin B. The hybridoma cells wereelectroporated as described previously (Lin et al., 1997), and selectedin 0.3 mg/ml hygromycin B. The ELISA spot assay was performed aspreviously reported (Zhang et al., 2001). Briefly, each drug resistantcolony was expanded to ˜1-5×10⁶ cells, and plated onto 96 well platesthat were pre-coated with anti-mouse IgM antibody. After 22 hours, theplates were developed for secreted IgM.

Constructs: Full length human AID (hAID) was amplified using primerspreviously reported (Zhang et al., 2001) and cloned into Zero BluntTOPOPCR Cloning Kit (Invitrogen) to sequence. The hAID insert was excisedwith EcoR1, blunted with Klenow polymerase, and cloned into pCEP4(Invitrogen) digested with PvuII. Vectors were digested with Nru1 andEcoRV prior to transfection.

PCR Amplification, cloning, and sequencing V- and C-regions regions fromcell lines: Genomic DNA was prepared as previously reported (Zhang etal., 2001). V-regions from the various B-cell lines were amplified withPfu polymerase (Stratagene) from genomic DNA using 30 cycles of 95°C./15 sec, 56° C./15 sec, 72° C./30 sec. Primers for N114 V-region, 5′primer: TTACCTGGGTCTATGGCAGT, 3′ primer: TGAAGGCTCAGAATCCCCC, and Cm2-3region 5′ primer: CCCCTCCTTTGCCGACATCTTCC, 3′ primer:TTCCATTCCTC-CTCGTCACAGTC. Primers for Ramos and P1-5 V-region werepublished before (Zhang et al., 2001, Sack et al., 2001). Primers forP1-5 Cg1 exons 2-3: 5′ primer: CACACAGCTCAGACGCAACCCC, 3′ primer:GGATCATTTACCAGGAGAGTGGGAGAGG. This primer pair amplifies both the Balb/cand the C57Bl/6 Cg1 segments from the P1-5 hybridoma, which can bedistinguished by allotypic differences. Since the NP-specific V-regionof P1-5 derives from C57Bl/6 (Tao and Bothwell, 1990), only C-regionssequences from C57Bl/6 are reported in FIG. 3 a. PCR products werecloned and sequenced as previously reported (Zhang et al., 2001).

Extraction of RNA, RT-PCR, and Northern Blots: ˜5×10⁶ cells were lysedwith 1 ml Trizol reagent (GibcoBRL) and RNA extracted according tomanufacturers instructions. ˜1 μg of total RNA was either run onformaldehyde gels for northern blots, or reverse transcribed using theSuperscript II kit (GibcoBRL). 5 μl of the RT product was diluted 5-foldwith H₂O sequentially 3 times. 1 μl of each of these 3 dilutions wasused in a PCR reaction, and all amplifications of each cDNA from eachdifferent clone were done together. Taq polymerase (Roche) was used toamplify GAPDH and AID using primers and conditions previously described(Zhang et al., 2001).

Statistics: Statistics for sequencing data in Table 2 and primary datafor reversion frequencies in FIG. 2 a were measured by theindependent-samples t-test with equal variances assumed (SPSS v.10).

Results

Three human B-cell lines (i.e. Ramos, BL-2 and CL-01) were recentlyshown to undergo SHM (Sale and Neuberger, 1998; Denépoux et al., 1997;Zan et al., 1999), thus opening the possibility of studing this processin vitro. In previous work (Zhang et al., 2001), we found that V-regionmutation rates in different Ramos clones correlated with the level oftheir AID mRNA, suggesting that AID plays an important role in SHM inRamos cells. Specifically, both the rates of mutation and the mRNAlevels of AID for Ramos clones 6 and 7 were higher than those for Ramosclone 1 ((Zhang et al., 2001; FIG. 1 a). To determine whether low AIDexpression per se was responsible for the low mutation rates in Ramosclone 1, this clone was stably transfected with either a vectorexpressing human AID (hAID) or an empty vector control. Mutation ratesof typical transfected clones were then determined by sequencingunselected V-regions after 1- or 2-months in culture (Table 1). Clonesexpressing low levels of AID (i.e. clones C.1 and A.1) had very fewmutations in the V-region, while clones that expressed ˜25 fold higherlevels of AID mRNA (i.e. clones A.2 and A.5) had many more V-regionmutations (FIG. 1 b and Table 1). Table 2 summarizes the mutationalfeatures of all the Ramos clones that expressed elevated levels of AIDand shows that the rates and characteristics of the mutations in all ofthese clones were similar: there was a targeting bias of G/Cnucleotides, transitions were slightly favored over transversions and˜35% of mutations were in RGYW (A/G, G, C/T, A/F) or WRCY hot spotsequences, motifs that are frequently targeted in SHM both in vivo andin vitro (Wagner et al., 1995; Rogozin and Kolchanov, 1992). These dataindicate that AID is required for SHM in Ramos cells. TABLE 1 V-regionmutations from cell lines transfected with empty vector (C) or hAIDconstruct (A)^(a) Mutation Mutated rates^(c) Frequency V- (mut/ Clone(months V-region Total bp (mut/bp) regions/ bp/gen) cultured)mutations^(b) sequenced ×10⁻⁴ total ×10⁻⁶ Ramos C.1 (1) 0 11200 <0.890/26 <2.5 Ramos C.1 (2) 1 12900 0.78 1/30 1.1 Ramos A.1 (2)^(d) 1 116000.86 1/27 1.2 Ramos A.2 (1) 5 12900 3.9 4/30 10.7 Ramos A.2 (2) 7 125005.6 7/29 7.7 Ramos A.5 (1) 6 16300 3.7 4/38 10.2 Ramos A.5 (2) 13 153008.5 9/31 11.8 P1-5 C.1 (2) 1 11900 0.84 1/35 1.4 P1-5 C.2 (2) 0 7140<1.4 0/21 <2.3 P1-5 A.1 (2) 28 28900 9.3 22/85  15.7 P1-5 A.2 (2) 6 578010.4 6/17 17.3 N114 C.1 (1) 0 10980 <0.91 0/18 <3.0 N114 A.3 (1) 1221960 5.5 11/36  18.2^(a)Duplicate mutations were counted only once, unless genealogiesindicate the mutation was unique.^(b)The V region corresponds to a 550 bp, 340 bp, and 610 bp region inRamos, P1-5, and N114 cells, respectively.^(c)Rates were calculated using a 20-, a 24-, and a 24-hour generationtime for Ramos, P1-5, and N114 cells, respectively.^(d)Expression of AID was low in this clone (FIG. 1b).

Ramos cells express surface markers that suggest that their normalcellular counterpart is a germinal center centroblast (Sale andNeuberger, 1998), which are the cells that normally undergo SHM.Although AID is required for SHM in these cells, other factors specificto centroblasts might also be required. To test this notion, wedetermined whether AID could induce SHM in hybridomas, which representplasma cells that are beyond the developmental stage that carries outSHM. We first examined the N89 and N114 hybridomas because they havenonsense codons within the V-region of their endogenous antibody heavychain gene (Connor et al., 1994; top of FIG. 2 a), allowing us to assaymany independently transfected clones by assaying for nonsense codonrevertants using the ELISA-spot assay (Zhang et al., 2001).

N89 and N114 cells were stably transfected with the hAID expressionvector and individual drug-resistant colonies were expanded and assayedfor secreted IgM with the ELISA spot assay. Each ELISA spot indicatedthat a cell had reverted the nonsense codon and was secreting antibody(Lin et al., 1997; FIG. 2 a inset). FIG. 2 a shows the frequency ofrevertants identified for each individual clone. None of the N89 andN114 clones that were transfected with the empty vector displayed arevertant frequency above 10⁻⁶ (FIG. 2 a). However, more than 50% ofindividual N89 and N114 clones transfected with the hAID construct hadrevertant frequencies higher than 10⁻⁶ (FIG. 2 a).

To determine the rate of mutation at the nonsense codon of N114, twelvesubclones of the AID-positive N114 A.3 clone were analyzed forrevertants (FIG. 2 a, right panel) yielding a mutation rate of 1.4×10⁻⁶mut/bp/gen, as calculated by fluctuation analysis (Zhang et al., 2001).As will be shown below, this reversion rate greatly underestimates themutation rate for the V region as a whole for two reasons: 1) thenonsense codon for N114 (and for N89) is not within an RGYW or WRCY hotspot motif, and 2) most mutations observed in these hybridoma cloneswere transition mutations in G/C nucleotides (see below) which wouldconvert the TGA nonsense codon to TAA, another nonsense codon.

Some N89 and N114 clones transfected with the hAID expression vector didnot revert at a detectable frequency (FIG. 2 a). Northern blots revealedthat all tested clones that did not express AID did not revert above10⁻⁶ (FIG. 2 b). However, some clones that expressed AID (i.e. N89 A.3,A.5, A.10, A.16, and N114 A.2, A.8; FIG. 2 b) also did not revert above10⁻⁶. While this suggests that AID does not induce SHM in all hybridomaclones, this is probably due to the inherently stochastic nature of thisanalysis: a nonsense revertant that arises early in the propagation of aclone will result in a culture that has accumulated revertants, while arevertant that arises late during the propagation of the clone will berepresented by very few revertants (Luria and Delbrook, 1943). This isexemplified by the high range of revertant frequencies in subclones ofN114 A.3, with some subclones having very low rates of reversion (FIG. 2a). This effect is expected to be more pronounced in the AID-transfectedN89 clones where mutation rates in the leader sequence (i.e. where thenonsense codon is located) are lower (Rada and Milstein, 2001; Rada etal., 1997).

To measure the real rate of mutation in these AID-expressing hybridomas,transfected clones were grown for 2 months, and unselected V-regionswere sequenced. A third hybridoma, P1-5 (Tao and Bothwell, 1990), wasincluded in this analysis because it expresses the same Vh186.2 heavychain V region that is used by C57BL/6 mice in their response to thehapten nitrophenyl (NP). This allows mutations to be compared betweenthis hybridoma cell and those found in vivo. Only the P1-5 and N114clones that expressed hAID mutated their V-regions (FIG. 2 b, Table 1).In both hybridomas, there were fewer mutations in the constant regionthan in the V region (1 and 3 mutations for P1-5 and N114, respectively;FIG. 3 a), showing that hypermutation was relatively restricted to theV-region. Furthermore, as suggested above, the mutation rates in thesehybridoma clones as calculated by sequencing (˜15×10⁻⁶ mut/bp/gen;Table 1) were ˜10 fold higher than those calculated by fluctuationanalysis of N114 A.3, as described above (1.4×10⁻⁶ mut/bp/gen; FIG. 2a). These overall mutation rates were similar to those induced by hAIDin Ramos clones A.2 and A.5 (i.e. ˜10×10⁻⁶ mut/bp/gen; Table 1). Thecharacteristics of mutations in the N114 hybridoma were also similar tothose seen in Ramos cells (Sale and Neuberger, 1998; Table 2). However,the types of mutations found in the V-region of the P1-5 clones weredifferent from the other cell lines in that mutations occurredexclusively in G/C nucleotides, transition mutations occurred at muchhigher frequencies than transversion mutations, and mutations weremostly found within RGYW and WRCY hot spots (Table 2, FIG. 3 b). Whilethis suggests that the mechanism responsible for A/T mutations is absentin the P1-5 hybridoma, which supports the two-phase model of SHM (Radaet al., 1998; Spencer et al., 1999), the high frequency of transitionmutations at G/C emphasizes the possibility that AID might be aDNA-specific cytidine deaminase. TABLE 2 Characteristics of mutationsobserved in Ramos clones 6 & 7, and hAID transfected cell lines. RamosRamos clones clones P1-5 clones N114 6 & 7^(a) A.2 & A.5 A.1 & A.2 cloneA.3 GC 40/51 (78%) 25/31 (81%) 34/34 (100%)^(d) 9/12 (75%) mutations/total T_(s) ^(b)/total 23/51 (49%) 13/31 (42%) 24/34 (71%)^(d) 7/12(58%) Deletions/  2/51 (4%)  2/31 (6%)  0/34 (0%) 0/12 (0%) total Hotspot^(c)/ 18/51 (35%) 10/31 (32%) 21/34 (62%)^(d) 6/12 (50%) total^(a)Previously published sequences (Zhang et al., 2001).^(b)T_(s): transitions (C to T or G to A)^(c)Mutations at underlined nucleotides within RGYW or WRCY are definedas hotspot mutations. 7% (36/550), 9% (32/340), and 6% (39/610) of totalnucleotides in V-regions of Ramos, P1-5 and N114, respectively, arehotspot nucleotides.^(d)Statistically significant when compared to Ramos clones A.2 & A.5 (p< 0.05 for GC mutations, p < 0.001 for T_(s) and for Hot spotmutations).Discussion

These findings show that AID is sufficient to activate SHM inplasma-like cells, indicating that its activity does not depend on othercentroblast-specific factors. We have previously used plasma-like NSOcells to measure SHM of immunoglobulin transgenes that contain V-regionnonsense codons (Lin et al., 1997). Sequencing unselected V regionsrevealed no mutations of the transgenes in 2 month-old culturesindicating low mutation rates in NSO cells. However, fluctuationanalysis for nonsense revertants revealed a wide distribution ofmutation rates (Id.) which depended strongly on the position of thenonsense codon in the V region, its proximity to hotspot motifs and thenumber of transgenes present in the transfectant. Of particularinterest, we did not detect expression of AID in NSO cells (data notshown). While we cannot rule out transient expression of AID in NSO, itis possible that the high rate of reversion which we observed in a fewof the NSO transfectants was engendered by the insertion site and/or thepresence of the transgene in a repetitive structure. Nevertheless, weestimate that the overall mutation rates in NSO are low compared toRamos clones and hybridomas expressing AID. Taken together with recentfindings that the pre-B-cell line 18.81 expresses AID and carries outSHM (Bachl et al., 2001), AID might be able to function at anydevelopmental B-cell stage, or perhaps even in non B-cells. This impliesthat AID functions alone, or with co-factors that are ubiquitouslyexpressed. In fact, many of the factors that have been shown to play arole in SHM, such as MSH2 (Rada et al., 1998; Phung et al., 1998;Reynaud et al., 1999; Vora et al., 1999), MSH6 (Wiesendanger et al.,2000), and DNA polymerases ζ (Zan et al., 2001) and η (Zeng et al.,2001; Rogozin et al., 2001) are enzymes expressed in most cells.

The findings reported here also have practical implications. The factthat hybridomas can be induced to undergo high rates of SHM withexpression of AID might allow one to obtain subclones that produceeither high-affinity monoclonal antibodies and/or antibodies that aremore specific. This has been a goal that has been sought by many (Korbinet al., 1990) since Kohler and Milstein first described the hybridomatechnology (Kohler and Milstein, 1975).

EXAMPLE 2 AID-Activated Somatic Hypermutation in Non-B-Cells

To test whether AID can activate SHM in non-B-cells, Bw5147 (a T-cellline) and CHO (a hamster ovary cell line) were used. These cells werefirst transfected with immunoglobulin heavy and light chain genes bystandard methods. Because the heavy chain gene (Igγ2a) has a nonsensecodon in the V-region, the ELISA-spot assay can be used to determinewhether AID can turn on SHM in these cells by assessing whether cloneshave reverted the nonsense codon and secrete IgG2a. Bw5147 and CHOclones that were stably-transfected with the immunoglobulin constructswere then transfected with empty vector or the vector expressing hAID.Individual drug resistant colonies were grown up and assayed by theELISA-spot assay. FIG. 4 shows that Bw5147 and CHO clones that expresshAID have statistically higher reversion frequencies than clones that donot express hAID. These data indicate that hAID can activate SHM innon-B-cells. In addition, because the transacting factors that normallyregulate immunoglobulin transcription are not present in thesenon-B-cells, cis-acting sequences that have been postulated to recruitthe mutator to the V-region of the immunoglobulin genes should not beactive. This indicates that hAID does not require tissue-specificcis-acting sequences to cause mutations, and further indicates that hAIDcan mutate any expressed gene.

EXAMPLE 3 Induction of Class-Switching by AID

In previous work, forced expression of AID in cultured B-cells (i.e.murine hybridomas and the human Burkitt's lymphoma cell line Ramos)turned on SHM with similar characteristics to that seen in vivo. Thissuggested that expression of AID was sufficient to initiate this processin B-cells that were at the wrong stage of differentiation. Since AIDhas also been implicated in class-switch recombination (Kinoshita andHonjo, 2001), we tested whether the Ramos clones that overexpress AIDcan be induced to class-switch to downstream isotypes. It is worthnoting that previous attempts to induce class switch recombination inRamos has either failed, or been only marginally successful. Since SHMis clonally unstable in Ramos cells (Zhang et al, 2001), and stabilityis due to the level of AID expression, we reason that Ramos failed toclass-switch in those previous experiments because clonally stable Ramoscells were being used that express low levels of AID.

To test this hypothesis, we stimulated the Ramos clones C.1, A.1 (lowAID; FIG. 5), and clones A.2, A.5 (high AID; FIG. 5) with CD40L(expressed on NIH3T3 cells) and IL-4. As controls, the same Ramos cloneswere incubated with NIH3T3 cells that were transfected withempty-vector. As illustrated in FIG. 5A, IgG was detected in thesupernatants of all clones, regardless of the level of AID, whenstimulated with CD40L and IL-4. However, IgG was also detected in thesupernatants of control-stimulated Ramos A.2 and A.5 (FIG. 5A),suggesting that they are hypersensitive to class-switching to the IgGisotype. To confirm these findings, we assessed whether mature IgGmessage can be identified in unstimulated and stimulated Ramos clones byRT-PCR While IgG mRNA (but not IgA or IgE, data not shown) can bedetected in all CD40L and IL-4-stimulated clones, IgG message is onlypresent in Ramos A.2 and A.5 in control stimulations (FIG. 5B). CD40Land IL-4-stimulation causes the production of Iγ3-sterile transcripts inBurkitt's lymphoma cell lines. As shown in FIG. 5B, Iγ3-steriletranscripts are present in all Ramos clones stimulated with CD40L andIL-4, but not in unstimulated clones A.2 and A.5 (FIG. 5B). Thissuggests that AID functions downstream in the induction of steriletranscripts. Collectively, these data indicate that AID expressionhyper-sensitizes Ramos cells to class-switch recombination.

EXAMPLE 4 Somatic Hypermutation of the AID Transgene in B and Non-BCells

This example was published as Martin & Scharff (2002b).

EXAMPLE SUMMARY

Expression of MD is sufficient to activate SHM in hybridomas, in non-Bcells, and in E. coli (See previous examples and Harris et al., 2002),suggesting that it initiates the mutational process by deaminating DNA.However, the cis-acting sequences that are responsible for targeting AIDactivity to the V-region of immunoglobulin genes are unknown. Here weconfirm that expression of AID in B cell lines (i.e. Burkitt's lymphomaRamos and hybridoma P1-5) not only causes hypermutation ofimmunoglobulin sequences, but also of other genes, in particular the AIDtransgene itself. Because it is possible that B cell-specifictransacting factors bind to and recruit the ‘mutator’ to the AIDtransgene, we tested whether the AID transgene can mutate in non-Bcells. Indeed, we show that expression of AID in chinese hamster ovary(CHO) cells causes SHM of both the immunoglobulin and AID transgenes.These data confirm that high transcription rates alone appear topredispose any gene to mutation by AID.

Introduction.

Mice and humans with mutations in activation-induced cytidine deaminase(AID) have defects in SHM (Muramatsu et al., 2000; Revy et al 2000),class-switch recombination (Muramatsu et al., 2000), and gene conversion(Arakawa et al., 2000; Harris et al., 2002). Based on the sequencesimilarity of AID to the RNA-editing enzyme APOBEC-1 (Muramatsu et al.,1999), it was postulated that AID might function by editing a specificmessage that results in the production of an altered protein thatsubsequently causes mutation (Kinoshita and Honjo, 2001). However, thehigher than expected number of transition mutations in V-regions(Golding et al., 1987) suggests that AID is a DNA-specific cytidinedeaminase that converts C to U nucleotides directly in the DNA of theV-region. In fact, we recently showed that AID induced the P1-5hybridoma to exclusively mutate G•C basepairs and most of thesemutations were transition mutations (Martin et al., 2002). With thefinding that AID can induce SHM in non-B cells (Yoshikawa et al, 2002)and in E. coli (Harris et al., 2000), it is more likely that AID is aDNA-specific cytidine deaminase.

The mechanism for targeting of SHM to the V region of immunoglobulingenes is not known. SHM has also been observed to occur in othernon-immunoglobulin genes. The genes for bcl-6 (Pasqualucci et al., 1998& 2001; Shen et al., 1998) and FasL (Mushen et al., 2000) undergo SHMwith similar characteristics to those observed in the V-region ofimmunoglobulin genes, albeit at a lower rate. It is possible that thesegenes share cis-acting sequences with the immunoglobulin locus that areresponsible for recruiting AID activity. More likely, B cell-specificcis-acting sequences do not exist, and targeting of SHM to certain genesis due to high transcription rates and possibly other non-specificfactors (Martin & Scharff, 2002a). In this regard, since theimmunoglobulin gene is one of the most highly transcribed genes in Bcells, it would be mutated at higher rates than other genes. However,other transcribed genes have not been found to mutate above the PCRerror rate in germinal center B cells (Pasqualucci et al., 2001; Storbet al., 1998a; Shen et al., 2000). Thus, the issue of whether SHM istargeted by B cell-specific cis-acting sequences is not completelyresolved. In this report, we confirm SHM of other transcribed genes in Bcells and non-B cells activated for SHM by expression of AID.

Materials and Methods.

Constructs: Full length human AID (hAID) cloned into the pCEP4 vector(Invitrogen) has been described before (Martin et al., 2002). The vectorwas digested with Nru1 and EcoRV prior to transfection. The Vn/ECMVγ2a-construct and the LK-construct were previously described (Lin etal., 1998; Zhu et al., 1995).

Cell lines, cell culture and transfection conditions: Ramos and P1-5hybridomas were grown as previously described. Chinese hamster ovary(CHO) Pro-5 cells (obtained from P. Stanley, Albert Einstein College ofMedicine) were grown in DME medium supplemented with 10% fetal calfserum. CHO cells were first electroporated with 10 μg of theLK-construct. One transfectant that secreted high levels of light-chain(CHO-LC 18) was then transfected with 10 μg of the mouse Vn/ECMVγ2a-construct. One of these transfectants (CHO-LC18-Vn/ECMV clone 8) wasthen transfected with 10 μg of the linearized hAID or the empty vectors.CHO cells were transfected in DME medium at 400 volts, 960 μF, platedinto 96-well plates, and selected with 1.5 mg/ml G418 for the Vn/ECMVγ2a-construct and 0.6 mg/ml hygromycin B for the hAID and emptyconstructs. The ELISA spot assay was performed as previously reported(Zhang et al., 2001). Briefly, each drug resistant colony was expandedto ˜1-5×10⁶ cells, and plated onto 96-well plates that were pre-coatedwith anti-mouse IgG2a antibody. After 20 hours, the plates weredeveloped for secreted IgG2a.

PCR Amplification, cloning, and sequencing V-regions and AID transgene:Genomic DNA was prepared as previously reported (Zhang et al., 2001).V-regions from the various B-cell lines were amplified with Pfupolymerase (Stratagene) from genomic DNA using 30 cycles of 95° C./15sec, 56° C./15 sec, 72° C./30 sec. Primers for AID, 5′ primer:5′GAGGCAAGAAGACACTCTGG3′, 3′ primer: 5′GTGACATTCCTGGAAGTTGC3′; bcl-6,5′primer: CCGCTCTTGCCAAATGCTTTG, 3′ primer: CACGATACTTCAT-CTCATCTGG;c-myc, 5′ primer: AGAAAATGGTAGGCGCGCGTA, 3′ primer:TCGACTCATCTCAGCATTAAAG PCR products were cloned and sequenced aspreviously reported (Zhang et al., 2001). Stratagene reports that PFUpolymerase has an error rate of ˜1/650,000 base pairs per duplication.Therefore, in a 30 cycle amplification, we expect ˜1 mutation in 20,000nucleotides to be attributed to PCR-error. Accession numbers for mutatedsequences of the AID gene for Ramos A.2 and A.5 is AF529815-AF529827,for hybridoma P1-5 A.1 and A.2 is AF529828-AF529840, and for CHO A.3 andA.9 is AF529841-AF529856.

Extraction of RNA and Northern Blots: ˜5×10⁶ cells were lysed with 1 mlTrizol reagent (GibcoBRL) and RNA was extracted according tomanufacturer instructions. ˜1 μg of total RNA was run on formaldehydegels for Northern blots.

Statistics: Statistics for primary reversion data in FIG. 7B wascalculated by the independent-samples t-test with equal variancesassumed (SPSS v.10). Results.

We previously showed that the Burkitt's lymphoma Ramos clone 1, whichdoes not undergo SHM and expresses low levels of AID, could be activatedfor V-region SHM by overexpressing AID (Martin et al., 2002).Specifically, the V-region (V) accumulates many mutations in Ramosclones A.2 and A.5 (Table 3) that have ˜25 fold higher levels of AIDmRNA than Ramos clones C.1 and A.1 (Id.). Bcl-6 and the c-myc allelethat has translocated into the switch region have also been shown toundergo SHM in B cell lines (Bemark and Neuberger, 2000; Zan et al.,2000). To determine whether overexpression of AID in Ramos cells alsoactivated mutation in these genes, bcl-6 and c-myc were sequenced fromtwo month-old cultures of Ramos clones that overexpressed AID. However,only a few mutations were found in the c-myc and bcl-6 genes in Ramosclones A.2 and A.5 (data not shown). RT-PCR analysis revealed that thesegenes were indeed being transcribed (data not shown). Because SHMcorrelates with transcription rates (Bachl et al., 2001), it is possiblethat the rates of transcription of these genes might be too low for SHMto occur at the level seen in the V-region. TABLE 3 Somatichypermutation of the V region and of the AID transgene in Ramos,hybridoma P1-5, and CHO cells. Level of AID Mutation Clone expression¹Frequency Mutated rates³ (months (vector Total bp (mut/bp) sequences/(mut/bp/gen) cultured) used) Mutations² sequenced ×10⁻⁴ total ×10⁻⁶Ramos AID^(low)  1 (V)⁴ 12900 0.78 1/30 1.1 C.1 (2) (empty vector) RamosAID^(low)  1 (V)⁴ 11600 0.86 1/27 1.2 A.1 (2) (sense hAID) RamosAID^(hi)  7 (V)⁴ 12500 5.6 7/29 7.7 A.2 (2) (sense  7 (A) 17400 4.0 6/295.6 hAID) Ramos AID^(hi) 13 (V)⁴ 15300 8.5 9/31 11.8 A.5 (2) (sense  8(A) 15600 5.1 7/26 6.9 hAID) Ramos AID^(low)  2 (A) 15020 1.3 2/29 3.6αA.1 (1) (anti-sense hAID) Ramos AID^(low)  1 (A) 8200 1.2 1/14 3.3 αA.2(1) (anti-sense hAID) P1-5 A.1 AID^(hi) 28 (V)⁴ 28900 9.3 22/85  15.7(2) (sense 10 (A) 14400 6.9 9/24 11.6 hAID) P1-5 A.2 AID^(hi)  6 (V)⁴5780 10.4 6/17 17.3 (2) (sense  7 (A) 6600 10.6 5/11 17.7 hAID) CHOAID^(hi)  6 (A) 12600 4.8 6/21 8.0 A.3 (2) (sense hAID) CHO AID^(hi) 11(A) 11400 9.6 11/19  16.0 A.9 (2) (sense hAID) CHO AID^(neg)  1 (A)10800 0.9 1/18 3.0 αA.1 (1) (anti-sense hAID) CHO AID^(neg)  1 (A) 94901.1 1/16 3.7 αA.5 (1) (anti-sense hAID)¹Expression levels of AID for Ramos clones was previously published (8).Expression of AID was negative (AID^(neg)), low (AID^(low)), or high(AID^(hi)).²Mutations identified in the V-region (V) = 430 bp, AID transgene (A) =600 bp.³Mutation rates were calculated using a 20-, a 24-, and a 24-hourgeneration time for Ramos, P1-5, and CHO cells, respectively.⁴Data previously published (8) from the identical clones and DNA samplesused to analyze AID mutations.

To test whether a highly transcribed non-immunoglobulin gene wasundergoing SHM in the same Ramos clones, we sequenced the highlytranscribed AID transgene that is regulated by the CMV promoter. Indeed,many mutations were identified within the AID transgene (A) in Ramosclones A.2 and A.5 (FIG. 6 and Table 3), and the calculated rates ofmutation were only slightly lower than that of the V-region (Tables 3and 4). In addition, the characteristics of the mutations in the AIDtransgene were similar to those in the V region (Table 4). Inparticular, 20% of all mutations occurred at G•C basepairs within RGYWor WRCY hot spot motifs, even though only 7% of G/C nucleotides withinthe AID transgene occur at these sequences (Table 4). RGYW and WRCYhotspot motifs are frequently mutated during SHM in vitro and in vivo(Rogozin and Kolchanov, 1992). To confirm that mutations observed in theAID transgene were due to the AID protein, the non-mutating Ramos clone1 was transfected with a construct in which the AID transgene is in theantisense orientation to transcription. In this case, only a fewmutations were found within the AID transgene (Ramos clones αA.1 andαA.2: (A); Table 3). To confirm that the hAID construct that was usedfor transfection did not contain mutations, AID was amplified from thehAID plasmid with PFU polymerase, cloned, and sequenced. Only onemutation was found at an A•T basepair in 10 clones (6000 nucleotides)sequenced. TABLE 4 Characteristics of mutations observed in the V regionand the AID transgene in Ramos, P1-5, and CHO cells. Ramos P1-5 CHO AIDAID AID V region transgene transgene transgene A.2 & A.2 & V region A.1& A.3 & Characteristic A.5¹ A.5 A.1 & A.2¹ A.2 A.9 Mutation 9.8 6.3 16.514.7 12.0 rates² GC 25/31 (81%) 8/15 (53%) 34/34 (100%) 15/17 (88%)11/17 (65%) mutations/total T_(s) ³ mutations/ 13/31 (42%) 8/15 (53%)24/34 (71%) 10/17 (59%) 11/17 (65%) total Hot spot⁴/total 10/31 (32%)3/15 (20%) 21/34 (62%)  8/17 (47%)  4/17 (24%)¹Data previously published (8).²Mut/bp/gen.¹Data previously published (8).²Mut/bp/gen.³Transition mutation (i.e. C to T, T to C, G to A, A to G)⁴G·C basepairs within RGYW/WRCY motifs are designated as hotspotnucleotides. 39/597 (7%) of nucleotides in AID transgene, and 7%(36/550) and 9% (32/340) of nucleotides in the V region of Ramos andP1-5, respectively, are hot spot nucleotides.

To determine if SHM of the AID transgene can also occur in other B celllines, we tested whether the AID transgene was being mutated in the P1-5hybridoma. This hybridoma is unique in that AID expression inducesmutations exclusively at G•C basepairs in the endogenous V region thatare mostly within RGYW/WRCY hotspot motifs (P1-5 clones A.1 and A.2;Table 4)(Martin et al., 2002). This suggests that this cell line ismissing a factor(s) responsible for the A•T mutations that are believedto occur during the second phase of SHM that is MMR- and polymeraseη-dependent (Rogozin et al., 2001; Zeng et al., 2001; Rada et al.,1998). Sequencing of the AID transgene in 2-month old cultures revealedmany mutations (FIG. 6 & Table 3). The calculated rates and frequenciesof mutation in the endogenous V region and in the ectopically-integratedAID transgene were similar (Table 3 and 4), and a striking bias formutations in RGYW/WRCY motifs was observed in both genes: 62% and 47% ofall mutations were within RGYW/WRCY motifs in the V-region and the AIDtransgene, respectively (Table 4). In addition, like in the V-region,most mutations occurred at G•C basepairs. The few A•T mutations in theAID transgene may have arisen by a non-AID related processes, such asduring the integration of the transgene into the genome (Wilkie andPalmiter, 1987). These data indicate that AID mutates both itself andthe immunoglobulin gene in B cell lines.

The data presented above suggest that hypermutation induced by AID doesnot require specific cis-acting sequences to localize mutation to aspecific gene. This is because the AID transgene is not expected toshare regulatory sequences with the immunoglobulin loci. However, it isformally possible that the hAID transgene and the CMV promoter-enhancerin particular contain sequences similar to the immunoglobulin loci thatare required for targeting SHM. It is also possible that the sites ofintegration that allow high expression of AID contain regulatorysequences that share motifs with the antibody gene. Because non-B cellsare not considered to have B cell-specific transacting factors,expressing the AID transgene in a non-B cell should cause any putative Bcell-specific cis-acting sequence to be silent. Thus, mutation of theAID transgene in non-B cells would argue against the requirement ofregulatory B cell-specific cis-acting sequences for targeting SHM. Wetherefore tested whether the AID transgene can mutate in non-B cells.

To confirm that human AID can activate SHM in chinese hamster ovary(CHO) cells, CHO cells were first stably transfected with murine heavyand light chain immunoglobulin genes (FIG. 7A). The murine heavy chainconstruct used in this experiment (i.e. Vn/ECMV γ2a-construct; FIG. 7A)has two unique features. First, the intronic μ enhancer was replacedwith the CMV enhancer to ensure that the immunoglobulin heavy chain genewas expressed in CHO cells. Northern blots confirm that the Vn/ECMVγ2a-transgene was expressed (data not shown). Second, a nonsense codonwas introduced into an RGYW hotspot in the variable region of the heavychain construct (FIG. 7A). This allows SHM to be measured by reversionof the nonsense codon that would result in the production and secretionof IgG2a that could be detected at the single cell level using theELISA-spot assay.

CHO cells stably expressing the murine immunoglobulin genes weretransfected with the hAID transgene, the antisense hAID transgene, andthe empty vector control. Independent transfectants were grown toapproximately 2×10⁶ cells, and distributed into ELISA plates coated withanti-murine γ2a antibody. After 20 hours, the ELISA plates weredeveloped for secreted antibody. The revertant frequency for eachindividual clone was then plotted (FIG. 7B, left panels). Because somehAID-transfected CHO cells did not express hAID (i.e. CHO clones A.4,A.8, A.13, A.16, A.17, A.19, A.21, FIG. 4), the revertant frequenciesfor each individual CHO clone was plotted in the relevant AID-negative(AID−) and AID-positive (AID+) columns (FIG. 7B). As shown in FIG. 7B(left panels), clones that express hAID reverted the nonsense-codon inthe Vn/ECMV γ2a-transgene ˜15 fold more frequently than clones that didnot express AID (p<0.01). To more accurately determine the mutationrates at the nonsense codon, 2 AID+ clones (i.e. CHO A.3 and A.9) weresubcloned. Ten subclones of each were assayed by the ELISA spot assay,and mutation rates were calculated by fluctuation analyses (19). CHOclones A.3 and A.9 displayed mutation rates of 4.4×10⁻⁶ and 5.0×10⁻⁶mutations per base pair per generation (mut/bp/gen), respectively (FIG.7B, right panels). Although these two clones chosen for further analysisinitially reverted at high frequencies (FIG. 7B, left panels), thecorresponding subclones displayed a similar range of reversionfrequencies and mutation rates to that of the larger group ofindependently transfected AID+CHO clones. It is unclear why CHO clonesthat do not express AID have such a high background of reversionfrequencies (AID−; FIG. 7B, left panel). Nevertheless, these datasupport findings (Yoshikawa et al., 2002) that AID can induce SHM innon-B cells.

To test whether the AID transgene was mutating in CHO cells, AID wassequenced from 2 month-old cultures of CHO clones A.3 and A.9. The AIDtransgene was found to contain many mutations in the sense (CHO clonesA.3 and A.9; FIG. 6 and Table 3) but not in the antisense orientation(CHO clones αA.1 and αA.5; Table 3). The calculated rates of mutation ofthe AID transgene were similar between the CHO clones and the B celllines (Table 4), and the characteristics of the mutations displayed asimilar pattern typical to SHM in cultured cells, namely a bias towardsmutations in G-C basepairs, a preference for transition mutations, andRGYW hot spot targeting (Table 4).

Discussion.

The work reported here indicates that the SHM process is not dependenton a specific cis-acting sequence(s) to target mutation to theimmunoglobulin gene, and will proceed with any cis-acting sequence thatconfers a high rate of transcription to the target gene. Two of ourfindings support this hypothesis: I) an immunoglobulin transgene mutatesin a non-B cell, and 2) the AID transgene driven by a strong promotermutates in B and non-B cells. Although it is possible that the ADtransgene has a B cell-specific cis-acting sequence(s), if this sequenceelement were to exist, it should be inactive in non-B cells since non-Bcells lack B cell-specific transacting factors. Similar findings tothose reported here were described in fibroblasts activated to mutate asubstrate when AID was overexpressed (Yoshikawa et al., 2002). The lackof a requirement of specific cis-acting sequences is also supported byfindings that other genes undergo SHM in B cells (Pasqualucci et al.,1998 & 2001; Shen et al., 1998; Muschen et al., 2000), and other genesessential for cell viability might also be mutated since constitutiveSHM appears to decrease the viability of cultured cells (Zhang et al.,2001). While the notion that SHM can occur in any highly transcribedgene is unsettling, this may explain why many types of lymphomas arisefrom B cells that are undergoing SHM (Pasqualucci et al., 2001; Kuppersand Dalla-Favera, 2001).

On the other hand, some observations support the notion that SHM isregulated by cis-acting sequences. First, other transcribed genes ingerminal center B cells do not undergo SHM (Pasqualucci et al., 2001;Storb et al., 1998a, Shen et al., 2000). The critical issue here iswhether these genes are in fact mutating, but at levels that are belowthe PCR error rate, or whether they are not mutating at all. Becausemutation rates are positively correlated with transcription rates (Bachlet al., 2001) and with RGYW/WRCY hot spot density (Michael et al.,2002), other genes might in fact be mutating, but at rates that simplycorrelate with the quantity of these other features. In this regard, theimmunoglobulin gene might be mutated at a higher rate than other genesbecause it is transcribed at very high rates. In addition, it must alsobe considered that accumulation of mutations downstream of promoterswill only occur in regions that do not confer a selective disadvantage,such as regions that do not contain open reading frames or regulatorysequences for housekeeping genes. Mutations in these regions shouldreduce the viability of the cell, and as a consequence, the apparentrate of mutation at these loci will seem to be low or absent.

Second, the classical observation that the V-region mutates at higherrates than the C-region also supports the idea that cis-acting sequencesare involved in targeting SHM. For example, a B cell-specific cis-actingsequence might affect the chromatin structure over the V-region allowingthe ‘mutator’ to gain access to the DNA. However, other explanationsexist that could account for this differential mutation of the V- versusC-regions without the requirement of B cell-specific cis-actingsequences. One possibility is that the ‘mutator’ associates with the RNApolymerase II complex to produce mutations during the initiation phase,but eventually falls off this complex during the elongation phase(Maizels, 1995; Storb et al., 1998b). Another possibility is thatmutation depends on the availability of single-stranded DNA (see below),and that there is more single-stranded DNA in the V-region than in theC-region. This in turn might be due to 1) stable RNA-DNA hybrids in theV-region as a result of transcription that leaves the non-transcribedstrand single-stranded, 2) a higher RNA polymerase II density in theV-region than in the C-region, or 3) transcription inducing stablesecondary DNA structures in the V-region with single-stranded loops ofDNA (Kinoshita and Honjo, 2001). While these models suggest thatmutation can be focused on the V-region without the requirement ofcis-acting sequences, there is presently no data to support thesebeliefs.

Many of the findings that support the notion that SHM is not regulatedby B cell-specific cis-acting sequences come from reports, includingthis one, where AID is overexpressed at levels that are believed to behigher than in centroblasts (Martin et al., 2002; Yoshikawa et al.,2002; Okawaki et al., 2002), although this value is not known. Becauseoverexpression of APOBEC-1, which is homologous to AID, resulted inhyper-editing of its target substrate ApoB mRNA (Davidson and Shelness,2000), it is possible that targeting of SHM has been deregulated incells that overexpress AID. Thus, caution must be exercised wheninterpreting this data. In addition, mutation rates induced byexpression of AID in cell lines are ˜10 fold lower than the ratesobserved in the V-region in vivo (Martin and Scharff 2002a). Thus otherB cell-specific factors might indeed help focus mutation over theV-region.

AID has been postulated to function directly (i.e. to directly deaminatecytidines in DNA [Kinoshita & Honjo, 2001; Martin et al., 2002]) andindirectly (i.e. via its putative mRNA editing activity [Kinoshita &Honjo, 2001]) in the SHM process. The fact that human AID can activateSHM in a hamster ovary cell line and in E. coli (Petersen-Mahrt et al.,2002) argues against AID having an indirect role in SHM since this wouldrequire that the transcript edited by AID be expressed ubiquitously andhave the same recognition motif in different species. Further support tothe notion that AID is a DNA-specific cytidine deaminase was provided bythe finding that AID predominantly caused transition mutations at G-Cbasepairs in the P1-5 hybridoma (Martin et al., 2002), in fibroblasts(Yoshikawa et al., 2002), and in E. coli (Petersen-Mahrt, 2002). Inaddition, the mutation rates induced by AID in E. coli is increasedslightly in the absence of uracil DNA glycosylase (Petersen-Mahrt etal., 2002) suggesting that uracil is an intermediate in the SHM process.Thus, AID might initiate SHM by deaminating cytidines on DNA resultingin the recruitment of the mismatch repair system and/or uracil DNAglycosylases (Martin and Scharff, 2002a; Petersen-Mahrt, 2002;Poltoratsky et al., 2000), which in turn could cause mutations at otherbasepairs during the repair phase. Since enzyme-catalyzed cytidinedeamination probably requires single-stranded DNA (because the aminogroup on cytidine is hydrogen-bonded to the carboxyl of guanosine), AIDmight therefore chose its target based on the availability ofsingle-stranded DNA (Martin and Scharff, 2002a).

The findings presented in this report also have practical implications.Because the AID transgene was found to mutate, it is likely that anytransgene under the regulation of a strong promoter will mutate as longas AID is expressed in that cell. Semi-random mutagenesis can facilitatethe characterization of the gene products for structure/functionanalysis.

EXAMPLE 5 Flanking a Gene with Foreign Sequences Inhibits SomaticHypermutation for that Gene

As shown in Example 4, the ectopically-integrated CMV-driven AIDtransgene (with a hygromycin-resistance selectable marker) undergoessomatic hypermutation in B and non-B cells. That is, AID mutates boththe immunoglobulin genes and itself. However, when a second vector (witha puromycin-resistance selectable marker), which had the CMV-driven AIDtransgene flanked by ˜2 kb vector-derived bacterial sequences, wasintegrated into N114 hybridoma cells, the AID gene did not undergosomatic hypermutation, even though immunoglobulin genes did mutate withthis AID transgene. This lack of somatic hypermutation of the AIDtransgene was not due to reduced transcription of the AID gene in thecells transfected with the second vector, because the AID transgene wastranscribed at the same rates in cells transfected with either vector.Thus the difference in mutation between the two vectors is not due totranscription differences, but is apparently due to the presence offlanking bacterial sequences in the second vector.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

1. A method of inducing a mutation in a gene in a eukaryotic cell,wherein the gene is operably linked to a promoter, and wherein the geneis within about two kilobases of the promoter, the method comprisingexpressing a transgenic activation-induced cytidine deaminase (AID) genein the cell.
 2. The method of claim 1, wherein the gene is also operablylinked to an enhancer.
 3. The method of claim 2, wherein the enhancer isan immunoglobulin enhancer.
 4. The method of claim 1, wherein the geneis between 10 bases and 2 kb in the 3′ direction from the promoter. 5.The method of claim 1, wherein the promoter is an immunoglobulinpromoter.
 6. The method of claim 1, wherein a polyA mRNA of the gene issynthesized in the cell, the polyA mRNA of the gene comprising at least0.01% of total polyA mRNA in the cell.
 7. The method of claim 6, whereinthe polyA mRNA of the gene comprises at least 0.1% of total polyA mRNAin the cell.
 8. The method of claim 6, wherein the polyA mRNA of thegene comprises at least 0.5% of total polyA mRNA in the cell.
 9. Themethod of claim 6, wherein the polyA mRNA of the gene comprises at least1% of total polyA mRNA in the cell. 10-12. (canceled)
 13. The method ofclaim 1, wherein the AID gene is flanked by a sequence foreign to thecell, wherein the sequence foreign to the cell is at least 200 bp long.14. (canceled)
 15. The method of claim 13, wherein the sequence foreignto the cell is at least 2000 bp long. 16-17. (canceled)
 18. The methodof claim 1, wherein the cell is a yeast cell.
 19. The method of claim 1,wherein the cell is a vertebrate cell.
 20. The method of claim 19,wherein the cell is a mammalian cell.
 21. The method of claim 20,wherein the cell is a B-cell.
 22. The method of claim 20, wherein thecell is a hybridoma.
 23. The method of claim 20, wherein the cell is ahuman cell.
 24. The method of claim 1, wherein the gene is an antibodygene.
 25. The method of claim 1, wherein the gene encodes a proteinselected from the group consisting of an enzyme, a transcription factor,a cytokine, and a structural protein. 26-33. (canceled)
 34. A method ofdetermining the effect of mutations in a gene encoding a protein on thephenotype of the protein in a eukaryotic cell, wherein the gene isoperably linked to a promoter, and wherein the gene is within about twokilobases of the promoter, the method comprising (a) expressing theprotein and a transgenic AID gene in the eukaryotic cell; (b)establishing clonal colonies of the cell; (c) identifying clonalcolonies that produce a gene of the protein that has a mutation; (d)determining whether the protein expressed by the mutated gene in anyclonal colony identified in step (c) has an altered phenotype; and (e)associating the altered phenotype with a particular mutation. 35-57.(canceled)
 58. A method of inducing a mutation in an antibody gene in aeukaryotic cell, the method comprising expressing a transgenic AID genein the cell. 59-96. (canceled)
 97. A method of inducing a class switchin an antibody heavy chain gene in a eukaryotic cell, the methodcomprising expressing a transgenic AID gene in the cell. 98-124.(canceled)
 125. A method of altering an affinity or a specificity of amonoclonal antibody to an antigen, or altering a cross-reactivity of themonoclonal antibody to a second antigen, wherein the monoclonal antibodyis produced by a eukaryotic cell, and wherein the cell is capable ofexpressing a transgenic AID gene under inducible control, the methodcomprising (a) expressing the AID gene in the eukaryotic cell for a timeand under conditions sufficient to induce a mutation in a gene encodingthe monoclonal antibody; (b) suppressing expression of AID gene in theeukaryotic cell; (c) establishing clonal colonies of the cell; and (d)determining whether the monoclonal antibody produced by any of theclonal colonies of the cell has altered affinity or specificity to theantigen, or altered cross-reactivity to the second antigen. 126-158.(canceled)
 159. A eukaryotic cell comprising a transgenic AID gene,wherein expression of the AID gene is inducible. 160-171. (canceled)172. The cell of claim 159, wherein the cell is a myeloma cell.
 173. Thecell of claim 159, wherein the cell is a hybridoma cell. 174-179.(canceled)
 180. A eukaryotic cell expressing an AID gene, wherein thecell is not a B-cell. 181-190. (canceled)
 191. The cell of claim 180,wherein the cell is a yeast cell.
 192. The cell of claim 180, whereinthe cell is a vertebrate cell.
 193. The cell of claim 192, wherein thecell is a mammalian cell.
 194. The cell of claim 193, wherein the cellis a human cell. 195-202. (canceled)
 203. A myeloma fusion partnerexpressing an AID gene. 204-212. (canceled)
 213. The myeloma fusionpartner of claim 203, wherein the fusion partner is selected from thegroup consisting of a Sp2/0-Ag 14, a FOX-NY, a P3X63, NX-1, a P3, aP3X643 Ag8.653, a NS1, and a NSO.
 214. A hybridoma expressing an AIDgene. 215-261. (canceled)